Download pdf - Species-Speciﬁc Differences in the Medial Prefrontal

Species-Specific Differences in the Medial PrefrontalProjections to the Pons Between Rat and Rabbit

Maria V. Moya,1 Jennifer J. Siegel,1* Eedann D. McCord,1 Brian E. Kalmbach,1 Nikolai Dembrow,1

Daniel Johnston,1,2 and Raymond A. Chitwood1

1Center for Learning & Memory, University of Texas at Austin, Austin, Texas 787122Department of Neuroscience, University of Texas at Austin, Austin, Texas 78712

ABSTRACTThe medial prefrontal cortex (mPFC) of both rats and

rabbits has been shown to support trace eyeblink con-

ditioning, presumably by providing an input to the cere-

bellum via the pons that bridges the temporal gap

between conditioning stimuli. The pons of rats and rab-

bits, however, shows divergence in gross anatomical

organization, leaving open the question of whether the

topography of prefrontal inputs to the pons is similar in

rats and rabbits. To investigate this question, we

injected anterograde tracer into the mPFC of rats and

rabbits to visualize and map in 3D the distribution of

labeled terminals in the pons. Effective mPFC injections

showed labeled axons in the ipsilateral descending

pyramidal tract in both species. In rats, discrete clus-

ters of densely labeled terminals were observed primar-

ily in the rostromedial pons. Clusters of labeled

terminals were also observed contralateral to mPFC

injection sites in rats, appearing as a less dense "mir-

ror-image" of ipsilateral labeling. In rabbits, mPFC

labeled corticopontine terminals were absent in the ros-

tral pons, and instead were restricted to the intermedi-

ate pons. The densest terminal fields were typically

observed in association with the ipsilateral pyramidal

tract as it descended ventromedially through the rabbit

pons. No contralateral terminal labeling was observed

for any injections made in the rabbit mPFC. The results

suggest the possibility that mPFC inputs to the pons

may be integrated with different sources of cortical

inputs between rats and rabbits. The resulting implica-

tions for mPFC or pons manipulations for studies of

trace eyeblink in each species are discussed. J. Comp.

Neurol. 522:3052–3074, 2014.

VC 2014 Wiley Periodicals, Inc.

INDEXING TERMS: PFC; corticopontine; anterograde tracer; pontine nuclei; corticocerebellar

An understanding of how the brain supports learning

and memory requires knowledge of the specific connec-

tivity between participating brain regions and the motor

systems that mediate and express learning. Trace eye-

blink conditioning is an example of associative learning

that requires an interaction between forebrain regions

and the cerebellum, via the pontine nuclei (Clark et al.,

2002; Kalmbach et al., 2009, 2010; Weiss and Dister-

hoft, 2011; Siegel et al., 2012). After many pairings of

a conditioned stimulus (CS, e.g., a tone) with an uncon-

ditioned stimulus (US, e.g., a puff of air to the eye ini-

tiating a reflexive eyeblink closure), animals learn that

the CS predicts the US and close the eyeblink in antici-

pation of the US. When the CS and US are separated

by a stimulus-free interval ("trace" eyeblink condition-

ing) the cerebellum relies on additional input from the

forebrain to bridge the temporal gap between stimuli to

learn and express conditioned responses. Rabbits and

rats are both able to learn this task, with lesion studies

indicating that both the medial prefrontal cortex (mPFC)

and cerebellum are necessary for the expression of

conditioned responses (Kronforst-Collins and Disterhoft,

1998; Takehara et al., 2003; Weible et al., 2003; Powell

et al., 2005; Kalmbach, 2008), and single-unit studies

in the mPFC revealing neural activity capable of bridg-

ing the trace interval (Takehara-Nishiuchi and McNaugh-

ton, 2008; Siegel et al., 2012). However, there are

some apparent differences in learning between the two

The first two authors contributed equally to this work.

Grant sponsor: National Institutes of Health (NIH); Grant numbers:MH094839 (to D.J. and M.D.M.), NS084473 (to D.J.), MH46904 andMH74006 (to M.D.M.); Grant sponsor: the McKnight Foundation.

*CORRESPONDENCE TO: Jennifer J. Siegel, Center for Learning & Mem-ory, University of Texas at Austin, 1 University Station Stop C7000, Austin,TX 78712-0805. E-mail: [email protected]

Received September 19, 2013; Revised January 18, 2014;Accepted February 18, 2014.DOI 10.1002/cne.23566Published online February 22, 2014 in Wiley Online Library(wileyonlinelibrary.com)VC 2014 Wiley Periodicals, Inc.

3052 The Journal of Comparative Neurology | Research in Systems Neuroscience 522:3052–3074 (2014)

RESEARCH ARTICLE

species. For example, most rats are unable to meet a

standard learning criterion (e.g., 60% response rate)

with a trace interval of 500 ms (Weiss et al., 1999;

Takehara et al., 2003; Takehara-Nishiuchi et al., 2006;

Nokia et al., 2012), while rabbits show robust learning

with trace intervals of 500 ms (Thompson et al., 1996;

Kalmbach et al., 2009, 2010; Siegel et al., 2012). The

neural basis for such differences is currently unknown,

but addressing such questions begins with an under-

standing of the species-specific differences in

prefrontal-pons-cerebellar projection patterns.

Previous work in rabbits has provided an initial map-

ping of the prefrontopontine projections that is the ana-

tomical basis for the expression of learning in the trace

eyeblink conditioning circuit (Buchanan et al., 1994;

Weible et al., 2007; Siegel et al., 2012). Our goal was

to extend the previous work in rabbits and to provide a

comparative mapping for the trace eyeblink condition-

ing circuit in rats. To this end, we made discrete injec-

tions of fluorescent dextran amines in regions of the

mPFC of rats and rabbits which previous work showed

was necessary for trace conditioning (compare Fig. 1 to

Takehara et al., 2003; Kalmbach et al., 2009), and

visualized labeled terminals in the pons. The antero-

grade tracer revealed species-specific differences in the

rostrocaudal extent of prefrontal terminals in the ipsilat-

eral pons, as well as the presence of projections to the

contralateral pons in rat that was distinctly absent in

rabbit. The results suggest that prefrontal inputs to the

pons may be integrated with different kinds of cortical

inputs between rats and rabbits, and that the rat pre-

frontal cortex may also support tasks that require bilat-

eral coordination of the cerebellum.

MATERIALS AND METHODS

Infusion of anterograde tracerAll procedures were approved by the University of

Texas at Austin Institutional Animal Care and Use Com-

mittee and were in accordance with the National Insti-

tutes of Health guidelines. The mPFC of rats and

rabbits were injected with a 10% solution of 10,000

MW dextran amines containing a fixable lysine residue

and conjugated to Alexa 488 or 594 (D22910 and

D22913, Invitrogen Molecular Probes, Grand Island,

NY), dissolved in artificial cerebrospinal fluid (in mM:

119.0 NaCl, 2.5 KCl, 1.2 NaH2PO4, 26.0 NaHCO3, 2.0

CaCl2, 2.0 MgCl2, 10.0 dextrose, 10.0 HEPES; pH

adjusted to between 7.35 and 7.4 and passed through

a 2-lm filter to sterilize). Each animal received at most

two injections in the mPFC: one at a caudal mPFC site,

and in most cases a second ipsilateral injection using a

different color fluorophore at a more rostral site. A

dedicated Hamilton (Reno, NV) syringe was paired with

a given fluorophore across surgeries to avoid any possi-

bility of contamination between injection sites.

Figure 1. Schematics showing anterograde tracer injection sites

within the mPFC of rats and rabbits. A: Representations of the

rat brain showing the locations of prefrontal injection sites (n 5 9

injections in seven animals, round markers) from dorsal (top right)

and lateral (bottom left) perspectives. Coronal representations

(top left) indicate structure at the most rostral and caudal injec-

tion sites (numbers indicate stereotaxic rostrocaudal coordinates

relative to Bregma). Location of the pons relative to the injection

sites is indicated in the lateral perspective (gray shaded region).

B: Representations of the rabbit brain showing the locations of

prefrontal injection sites (n 5 7 injections in five animals), as

described for rats in A. C: Example coronal sections from along

the rostrocaudal axis of mPFC (numbers indicate stereotaxic

coordinates relative to Bregma). Cumulative extent of layer V tis-

sue containing labeled somata across all cases for rat and rabbit

is shown in red. Boxplots (center) show the COM of labeled pre-

frontal tissue (black markers) and IQR (gray box) for rats and rab-

bits. Note that 75% of labeled prefrontal layer V tissue overlaps

in the rostrocaudal axis, and was injected in anatomically similar

prefrontal regions.

Species-specific MPFC projections to the pons

The Journal of Comparative Neurology | Research in Systems Neuroscience 3053

RatsMale Sprague–Dawley rats (n 5 8, 250–400 g; Charles

River Laboratories, Wilmington, MA) were anesthetized

with isofluorane (4% induction followed by 1–3% mainte-

nance). Each animal was placed in a stereotaxic appa-

ratus and the skull leveled based on the dorsal-ventral

coordinates of Bregma and Lambda (Paxinos and Wat-

son, 2007). Craniotomies were made over the target

regions of mPFC (between 1 and 5 mm anterior to

Bregma, 1 mm lateral to the midline; Figs. 1–3).{FIG1–

3} After the dura was retracted, a 1 lL Hamilton

syringe (model 7001KH, Fisher-Scientific, Pittsburgh,

PA) was filled with 0.25 lL of dextran tracer, and the

syringe tip lowered 1.5 mm below the surface of the

brain and allowed to settle for 10 minutes before begin-

ning manual pressure injection. Each animal received

two ipsilateral injections, one at a rostral location (3.0–

5.0 mm rostral to Bregma) and a second using a differ-

ent color fluorophore at a more caudal coordinate (1.0–

2.5 mm rostral to Bregma). Tracer was pressure

injected over 5 minutes (0.05 lL/min). The syringe was

left in place for a minimum of 10 minutes after injec-

tion to allow for diffusion and then withdrawn from the

brain. After injections were complete the craniotomies

were filled with Kwiksil (World Precision Instruments,

Sarasota, FL) and the skin of the animal sutured. Rats

survived 5–7 days before sacrifice to allow the antero-

grade tracer to be transported down the axons and fill

terminals in the pons.

RabbitsNew Zealand albino male rabbits (n 5 6, 3–4 kg; Myr-

tle’s Rabbitry, Thompsons Station, TN) were initially

anesthetized with 45 mg/kg ketamine mixed with 1.5

mg/kg acepromazine and placed in a stereotaxic appa-

ratus with Lambda 1.5 mm below Bregma (McBride and

Klemm, 1968). Anesthesia was maintained with 1–3%

isoflurane throughout surgery. A craniotomy was made

over the target region(s) of mPFC (between 1 and 5

mm rostral to Bregma, 1 mm lateral to midline; Figs. 1–

3). After the dura was retracted, a 1-lL Hamilton

syringe was filled with 1 lL of tracer and the syringe

tip lowered 1.5 to 2.0 mm below the surface of the

brain and allowed to settle for 10 minutes before man-

ual infusion of tracer. Anterograde tracer was pressure

injected over a 20-minute period (0.05 lL/min), after

which the syringe was left in place for 10 minutes

before being withdrawn. Each rabbit received an injec-

tion at a caudal mPFC location, and three of the rabbits

received a second injection using a different fluoro-

phore at a more rostral location. Craniotomies were

filled with Kwiksil and the skin of the animal sutured.

Rabbits survived for 7–9 days postsurgery.

Tissue collection and processingAnimals were given lethal injections (rats: ketamine,

160 mg/kg mixed with xylazine, 16 mg/kg, intraperito-

neal; rabbits: Euthasol 0.3 mL/kg, intravenous) and per-

fused intracardially with cold, 0.9% physiological saline

or oxygenated modified artificial cerebrospinal fluid

(aCSF) (2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM

NaHCO3, 0.5 mM CaCl2, 7 mM MgCl2, 7 mM dextrose,

205.5, mM sucrose, 1.3 mM ascorbic acid, and 3.7 mM

pyruvate), followed by 4% paraformaldehyde in 0.02 or

0.1 M phosphate buffer (pH 7.4). Brains were postfixed

for at least 2 days and then transferred in a 30%

sucrose solution until equilibrated for cyroprotection.

Tissue was sectioned using a sliding microtome (Leica

Microsystems, Buffalo Grove, IL) equipped with temper-

ature controlled freezing stage (Physitemp, Clifton, NJ).

Coronal sections of the mPFC (50–100 lm) and the

pons (50 lm) were transferred to 0.9% physiological

saline and mounted on Microfrost Plus slides (Fisher

Scientific). Mounted sections were protected from light

and dust, and air-dried overnight. Prior to coverslipping,

sections were washed in 50% EtOH (1 minute), followed

by 100% EtOH (1 minute), and then cleared in xylenes

for 10 minutes and coverslipped with DPX mounting

medium (Electron Microscopy Services, Hatfield, PA).

Image acquisition and processingImages of mPFC and pons sections were acquired

using a Zeiss Axio Imager Z2 microscope running Axio-

Vision software (v. 4.8.2; Carl Zeiss, Thornwood, NY).

Multichannel images were acquired and included up to

three independent reflected wavelength bands (blue,

green, and red) and one transmitted channel using dif-

ferential interference contrast. The fluorescent channels

included: one optimized for viewing the nuclear stain

40,6-diamidino-2-phenylindole dilactate (DAPI) and used

here to visualize autofluorescence of gross anatomical

structure (blue); another for viewing the fluorophore

Alexa-488 (green); and a third for viewing the fluoro-

phore Alexa-594 (red). In the case of conventional fluo-

rescence, standard Zeiss filter sets (set 49: ex G 365,

dc 395, em 445/50; set 38: ex 470/40, dc 495, em

525/50; set 71: ex 592/24, dc 615, em 675/100; for

blue, green, and red, respectively) were used to sepa-

rate the excitation/emission wavelengths. Little or no

crosstalk between channels was observed (e.g., Fig.

4A–C). Images were acquired as multichannel mosaics

in 12- or 16-bit grayscale format. Following alignment

of tiled images and assembly into a single continuous

image using the AxioVision software, individual channels

were exported as unmodified 8- or 12-bit TIF files.

A subset of images were reacquired using a Leica

SP5-RS 2-photon laser-scanning microscope running the

M.V. Moya, J.J. Siegel et al.

3054 The Journal of Comparative Neurology |Research in Systems Neuroscience

Leica Application Suite Advanced Fluorescence soft-

ware and using Dodt contrast enhancement. When 2-

photon excitation was used, the excitation wavelength

was set to 840 nm and the reflected green and red sig-

nals separated using standard filters (dichroic: DXCR

565; green bandpass: HQ 525/50; red bandpass: HQ

Figure 2. Identification of labeled layer V somata at prefrontal injection sites. A,B: Inverted autofluorescent (blue channel) images showing

delineation of neocortical cell layers in medial prefrontal cortex based on cell morphology (dashed yellow lines). Closed arrows show iden-

tified pyramidal cells (one is shown at higher magnification in B, autofluorescence not inverted here), open arrow shows example of gliotic

tissue. Boxes 1–3 show the corresponding subregions at higher magnification. C: Fluorescent image (right) overlaid over inverted autofluor-

escent image (left) used to identify neocortical layers. D: Fluorescent image with outlines of cortical surface and corpus callosum (solid

yellow lines) and neocortical cell layers (dashed lines), and outline of labeled layer V tissue (red line) and gliosis (gray line) for a single cor-

onal section. Line drawings show identified labeled tissue (red) and regions of gliosis (gray) at more anterior (left) and posterior (right) cor-

onal sections. E: Three-dimensional rendering of all prefrontal sections from this case showing the overall 3D structure of labeled layer V

tissue (far left), also shown from three additional rotated perspectives as indicated by directional compasses (left to right). A, anterior; P,

posterior; D, dorsal; V, ventral; M, medial; L, lateral. Filled gray arrow indicates region of gliotic tissue.



645/100) in an external, non-descanned detector. In

rare instances where the fluorescent signal was weak,

both the reflected and transmitted fluorescent emission

was collected to increase the signal-to-noise ratio.

Images from both conventional and two-photon tech-

niques were processed using ImageJ (NIH, http://

rsbweb.nih.gov/ij) or FIJI (Schindelin et al., 2012).

Unprocessed image stacks were imported using Bio-

Formats import utilities (Open Microscopy Environment,

http://openmicroscopy.org; Linkert et al., 2010).

Images were adjusted to maximize the distribution of

pixel values in an effort to increase contrast and mini-

mize background autofluorescence in the green and red

channels. Because the smallest processes were some-

times difficult to resolve against the background tissue

autofluorescence, the autofluorescence of a second

channel was subtracted from the original to enhance

the signal-to-noise ratio (Fig. 4A–C). Care was taken to

ensure the background autofluorescence was equivalent

in each channel—the mean fluorescence was measured

in an area devoid of labeled tissue and the images

adjusted to normalize the level of background across

green and red channels. Adjusted images were always

compared to their respective unaltered source files to

ensure the process eliminated the appropriate amount

of autofluorescence from the original image. This

method worked equally well for subtracting autofluores-

cence from either the green or red channel images.

Because the resulting image lacked information regard-

ing gross morphology, the autofluorescence from the

blue channel was optimized and inverted for the sole

purpose of visualizing the overall anatomical structure

of a given section.

Localization of mPFC injection sitesFluorescent images of all mPFC sections were initially

acquired at 2.53 magnification to view the general

infusion location, and again at 103 to determine the

extent of labeled neurons across mPFC subregions

and cortical layers (Fig. 2A–D). An anterior-posterior

stereotaxic coordinate relative to Bregma was

assigned to each section by first designating the sec-

tion containing the genu of the corpus callosum as a

reference point according to the atlases of Paxinos

and Watson (2007; rats, 12.30 mm from Bregma; Fig.

3 left panel) or McBride and Klemm (1968; rabbits,

13.25 mm from Bregma; Fig. 3 right panel) and

assigning an anterior-posterior coordinate to subse-

quent sections rostral and caudal to that point based

on section thickness.

Figure 3. Three-dimensional rendering of all medial prefrontal injection sites in rat (A) and rabbit (B). Different colors represent different

individual cases for each species. Numbers represent anterior-posterior stereotaxic coordinates relative to Bregma (B), directional com-

passes are as given in Figure 2. Note that a similar relative span of the medial prefrontal cortex was sampled in the two species.



http://rsbweb.nih.gov/ij

http://rsbweb.nih.gov/ij

Figure 4. Identification of clusters of putative prefrontal axon terminals and relative terminal density assignments. A–C: Example of

autofluorescence-subtracted processing of pons images. To ensure that even lightly or sparsely labeled terminal clusters would be detected

(example regions of light, box 1, and sparse, box 2, are given in A), relative background autofluorescence was normalized between the red

and green channels, and then the normalized background fluorescence (determined from areas where no labeling was ever observed) was

subtracted from the channel of interest resulting in readily identifiable axons and terminals (C, right). Note that there was no apparent cross-

talk in fluorescent signals between the red and green channels (B). D: Autofluorescence-subtracted pons image with outlines of the pontine

gray (Pons) and pyramidal tract (py) in yellow, and identified terminal clusters outlined in red. E: Cluster outlines were filled and opacity

adjusted to reflect the relative density of punctate terminals observed (ranging from 100% being the most dense observed in that animal to

25%). F,G: Examples of the density assignments before (F) and after (G) autofluorescence-subtraction, from example regions outlined in D,E.



Localization of pons labelingPons tissue was imaged at 103 magnification. The

rostral-caudal coordinates of pons sections were deter-

mined by assigning an anterior-posterior coordinate of

the most rostral section of the pons based on gross

anatomical features according to the atlases of Paxinos

and Watson (2007) or McBride and Klemm (1968) and

assigning an anterior-posterior coordinate to subse-

quent sections caudal to that point based on section

thickness.

AnalysisInclusion of data for analysisEight rats each received two injections targeted to sep-

arate locations along the rostral-caudal axis of the

mPFC. Of the 15 mPFC injections in the rat (one injec-

tion fell outside of the mPFC and was excluded), nine

injections resulted in clearly labeled axons in the

descending pyramidal tract ipsilateral to the injection

site (Fig. 4 shows an example of such labeling), indicat-

ing effective uptake and transport of the dextran by

mPFC layer V neurons. These nine cases were included

for further analysis (Figs. 1A,C, 3A). The six remaining

cases were either devoid of labeled axons in the pyram-

idal tract (n 5 5 cases) or showed very sparsely labeled

axons (n 5 1 case) relative to the extent of labeling typ-

ically observed. Double-labeled axons in the pyramidal

tract or pons were not observed, suggesting that there

was no diffusion of tracer across injection sites.

Labeled axons were never observed in the contralateral

pyramidal tract (e.g., Fig. 4).

Six rabbits received anterograde tracer injections in

the mPFC (four rabbits received two injections, as

described for rats, and two rabbits received a single

more caudal injection). The data from the two rabbits

with single caudal injections have been previously

reported (Siegel et al., 2012). In 7 of 10 experiments

labeled axons were observed in the ipsilateral descend-

ing pyramidal tract and were included for analysis (loca-

tions shown in Figs. 1B, 3B). The remaining three cases

resulted in minimal labeling of layer labeling of axons in

the pyramidal tract and no terminal labeling observed

in the pons. Similar to rats, labeled axons in the pyram-

idal tract contralateral to the injection site were never

observed, and no double-labeling was present.

The corpus callosum is located immediately adjacent

to the mPFC in rats and rabbits (e.g., Fig. 2E, CC in left

panel). This dense bundle of axons was largely undis-

turbed by the insertion of the syringe during dextran

injections in all cases. Although in some cases tracer

did diffuse to the corpus callosum, no evidence of

uptake directly by corpus callosum axons of passage

was observed (similar to previous observations; Glover

et al., 1986; Schmued et al., 1990). Labeled axons

were observed to enter the corpus callosum, typically

from the shoulder cortex area of mPFC, and were also

observed exiting the corpus callosum at the same loca-

tion in the contralateral hemisphere. No labeling of

soma in the contralateral mPFC was ever observed. The

contralateral projection between subregions of mPFC

has been previously described and was not analyzed

here (Sesack et al., 1989; Buchanan et al., 1994; Wei-

ble et al., 2007)

Restriction of mPFC injection sites to tissuecontaining labeled layer V neuronsIt was previously shown that mPFC projections to the

pons originate exclusively from layer V neurons (Legg

et al., 1989). We therefore restricted our injection site

measurements to dextran-labeled layer V cells.

Images of mPFC sections were imported as TIF files

into the serial-section reconstruction software Recon-

struct (http://synapses.clm.utexas.edu; Fiala, 2005)

and aligned using the midline and the dorsal surfaces

of each section as anchors. The boundaries of layer V

in all mPFC sections were determined by comparing

neuron somatic morphology readily discerned in the

inverted blue-channel image (e.g., Fig. 2A,B). The identi-

fication of cortical layer boundaries based on differen-

ces in neural morphology allowed for the inclusion of

layer V labeled tissue even if displaced by mechanical

insertion of the injection syringe. The "Point-by-Point

Tracing Tool" within Reconstruct was used to outline

regions of layer V tissue containing dextran labeled

neurons (e.g., Fig. 2D), which also gave the surface

area and 2D centroid (used to calculate the center-of-

mass of the labeled tissue; see below). We did not

include layer V tissue showing evidence of gliosis, often

associated with mechanical placement of the syringe

during the injection. In regions of gliosis, the blue-

channel autofluorescence image revealed an abundance

of small cell bodies that did not show neuronal mor-

phology (e.g., Fig. 2A, open arrow), and were easily

identified. The restriction of labeled tissue to layer V

and the exclusion of regions of gliosis resulted in irregu-

larly shaped masses of mPFC labeled layer V cells (Fig.

2D,E). The volumes of labeled layer V tissue outlined in

all sections associated with a given injection site (given

by the surface area of the defined boundary times slice

thickness) were used to create a 3D representation of

each Alexa-dextran injection (e.g., Figs. 2E, 3). It should

be noted the volume of labeled tissue would have been

underestimated if any removal of dextran from the

soma of neurons at the injection site occurred over

time.



Identification of the center of mass of mPFCinjection sitesThe medial-lateral, dorsal-ventral, and rostral-caudal

coordinates of the center of mass (COM) of injection

sites were calculated independently using the x or y

coordinates from the 2D centroids calculated for each

mPFC section (described above), or the z coordinates

given by the anterior-posterior stereotaxic coordinate of

the mPFC sections, using the following formula:

COMx; y or z5

Xn

i51rai

3SAi

SAT

where COM is the x, y, or z coordinate of the 3D cent-

roid. SAi indicates the surface area of layer V within

section i that contained labeled neurons and ra is

defined by the x, y, or z value for section i. SAT is the

sum of all labeled layer V surface areas identified in n

sections. The total volume of labeled layer V tissue was

also calculated by multiplying SAT by section thickness.

Note that the formula assumes a uniform density of

labeled neurons in calculating the COM of mPFC injec-

tion sites. The injection site COM and volume calcula-

tions reflect the overall extent of labeled tissue and

was insensitive to the density of labeled cells.

The cumulative interquartile range (IQR) of the mass

of each reconstructed 3D injection site was calculated

to describe the spread of labeling in layer V tissue

along the rostral-caudal axis. The quartiles were deter-

mined by multiplying the total mass by 0.25 to deter-

mine the 25th percentile, and by 0.75 to determine the

75th percentile. The mass from each section was then

cumulatively added in rostral-to-caudal order until the

25th and 75th percentiles of the mass were exceeded.

The stereotaxic coordinate of the corresponding sec-

tions that satisfied the quartiles was used as the IQR,

which describes the rostral-caudal range containing

50% of the mass of labeled layer V tissue—the larger

the IQR, the greater the spread of labeled tissue across

the rostral-caudal axis.

For each species, the injection sites were accumu-

lated across animals to compare total labeled mPFC tis-

sue between rat and rabbit. The rostral-caudal COM

was calculated using the same equation as above, with

SAi given as the sum of surface areas of identified

labeled tissue from all injections at given sections from

the same stereotaxic coordinate and SAT equal to the

sum of all surface areas for all injection sites. The

same procedure used to calculate the IQRs was applied

to the group summed mPFC injections as well.

In addition to calculating the COM, the extent of the

injections was examined to determine which mPFC sub-

regions showed labeled layer V tissue. The subregional

boundaries of mPFC (medial agranular, AGm; anterior

cingulate cortex, AC; prelimbic cortex, PL; infralimbic

cortex was not investigated here) were estimated

according to differences in laminar morphology (as

described by Terreberry, 1987). Labeled layer V tissue

that extended beyond the estimated boundary was con-

sidered to have labeled that subregion.

Identification of mPFC terminal labeling in thepons and center of mass calculationAfter acquiring and processing images of pons tissue, dis-

crete clusters of labeled terminals within each section

were identified using ImageJ or FIJI. The boundaries

around each discrete cluster of labeled terminals in each

pons section were defined using an outline tool, and the

area of each identified cluster calculated by converting

the number of pixels enclosed with the boundary to area

in lm2. The density of labeled terminals within discrete

clusters varied from relatively dense to fairly sparse, even

within the same pons section (e.g., Fig. 4E–G). Therefore,

density coefficients of 1.0, 0.75, 0.5, or 0.25 were

assigned to each cluster, with 1.0 equivalent to the most

dense clustering of labeled terminals observed for a given

animal, and 0.25 representing relatively sparse density

(e.g., Fig. 4E; Jones et al., 2005; Weible et al., 2007).

Because light scatter from dense terminal clusters out-

side the focal plane may have obscured individual presyn-

aptic elements, and to verify that the assigned cluster

density was not erroneously influenced by unfocused flu-

orescence, a subset of sections were examined using 2-

photon imaging techniques and validated the assign-

ments given using conventional fluorescent microscopy

(compare Fig. 4F to 4G). For illustrative purposes, the

assigned density coefficients were represented in figures

by the opacity value of the represented cluster bounda-

ries (100, 75, 50, and 25%), where an opacity of 100%

indicates a density coefficient of 1.0, and less opaque

regions indicate density coefficients proportional to the

level of opaqueness (e.g., Fig. 4E). Occasionally, relatively

large and highly irregular clusters were observed that

appeared to have nonuniform densities across the area,

but nevertheless appeared to be a continuous cluster. In

these atypical cases the cluster was divided in accord-

ance with density changes into two or three clusters and

the appropriate density coefficient assigned.

For each injection site, the COM of pons terminal

labeling in the rostral-caudal axis was calculated

according to the following equation:

COMz5

Xn

i51ri3SAi3qiXn

i51SA

i3qi

where, ri is the estimated rostral-caudal coordinate of

the section in which terminal cluster i is found, SAi is



the surface area of the terminal cluster in the section

and qi is its density coefficient. The denominator repre-

sents the total mass of the 3D cluster of terminals. The

IQR of terminal labeling in the pons was calculated for

each injection site using the same procedure as applied

to labeled layer V mPFC tissue. To quantify the overall

extent of labeling in a way that also accounted for the

density of labeling without excluding sparse inputs, we

weighted them according to assigned density coeffi-

cients (i.e., the area of each cluster was multiplied by

its density coefficient). Although a minimum designation

of 0.25 was used, sparse clusters tended to be rela-

tively small and thus did not have a substantial impact

on the results. Also, similar to the procedure for mPFC

injection sites, the terminal clusters observed for all

cases for a given species were collapsed to allow for

grouped summary comparisons in mPFC corticopontine

labeling between rat and rabbit, and the COMs and

IQRs calculated as described above.

3D representationsThe mPFC and pons were reconstructed from serial

images of Nissl-stained coronal sections (50 lm) using

custom software (Reconstruct, http://synapses.clm.u-

texas.edu; Fiala, 2005). The serial sections of a given

sample were aligned according to the structural fea-

tures in consecutive sections. For each section of

mPFC, the cortical surface, corpus callosum, and layer

V were manually outlined (e.g., Fig. 2C,D). Layer V tis-

sue that contained fluorescent dextran-labeled somata

was also manually outlined if present (Fig. 2D). For

each section of pons, the pontine gray and pyramidal

tracts were manually outlined (e.g., Fig. 4D,E). Clusters

of labeled putative axon terminals were also outlined

and filled with an arbitrary color that was adjusted in

opacity between 25–100% according to the designated

cluster density (0.25–1.0, as described above; e.g., Fig.

4D,E). The serial outlines of each sample of mPFC and

pons were rendered as "Boissonnat" 3D surfaces

(polygonal faces were automatically constructed to

make the outline points of consecutive sections contig-

uous). Rendered 3D images were exported as VRML

2.0 files and imported to Blender (http://www.blender.

org) for further processing. In Blender, the surfaces of

3D objects were passed with a "Smoothing Modifier"

function (smoothness factor of 1) to remove jagged ver-

tices and surface inconsistencies. The result was a

smoothed object that maintained the dimensions of the

original, unsmoothed object. Blender was also used to

assign case-specific colors to labeled tissue. The

appearance of punctate axon terminals in 3D pons

reconstructions were assigned arbitrarily by applying a

"Particle System" for each outlined cluster that was

composed of spheres with a density coefficient consist-

ent with the density assigned to a given cluster. The

spheres of particle clusters were also assigned case-

specific colors.

RESULTS

The mPFC of rats and rabbits was injected with

anterograde tracer to identify the projection pattern of

mPFC layer V neurons to the pons for each species.

For each injection site, the COM of tissue containing

dextran-labeled layer V neurons was calculated to more

accurately describe the location of the mPFC injection

in a stereotaxic coordinate frame. The area and respec-

tive density of labeled terminals resulting from each

injection was then determined and the combined

results were mapped onto a series of representative

PFC and pons sections and a 3D model constructed

from these sections.

mPFC injection sitesRatsThe sites of tracer injections in the rat (n 5 9) were

located 0.5–1.5 mm lateral to the midline, and were

distributed between 11.35B and 14.8B (mm anterior

to Bregma; Fig. 1A). While fluorescent tracer was often

observed across multiple layers of mPFC, diffusing both

outward from the base of the syringe and along the

entire syringe tract, it has been demonstrated that cor-

ticopontine projections arise exclusively from neurons

in layer V (e.g., Legg et al., 1989). Therefore, the extent

and precise location of labeled mPFC corticopontine

neurons was restricted to the volume of layer V tissue

in which brightly labeled cells were observed (Fig. 2,

see Materials and Methods). Regions of obvious gliosis

presumably resulting from mechanical damage associ-

ated with syringe insertion could be readily identified

and excluded from labeled tissue (e.g., Fig. 2A,D). This

likely contributed to irregularly shaped layer V injection

sites spanning 200–600 lm across the anterior-

posterior extent (median anterior-posterior extent of

labeling 5 500 lm; e.g., Figs. 2D,E, 3A). Six of the nine

injections spanned the entire extent of layer V (e.g.,

Fig. 2C,D). Although the volume of labeled layer V tis-

sue was relatively restricted, a given injection often

labeled more than one subregion of mPFC (6/9 cases),

based on relative anatomical location according to the

atlas of Paxinos and Watson (2007). The most common

sampled mPFC subregion was the medial agranular cor-

tex (AGm; also referred to as M2, Fr2, or PCm), labeled

in 7/9 cases ranging from 14.8B to 11.35B. The ante-

rior cingulate cortex (AC) was labeled in 6/9 cases but

within a more restricted anterior-posterior range than



http://www.blender.org

http://www.blender.org

AGm (14.3B and 12.4B). The prelimbic subregion (PL)

was also labeled in 6/9 cases (only the dorsalmost

aspect at the presumed AC border) ranging between

14.8B and 12.3B (to the genu of the corpus callosum

and the caudalmost extent of that subregion). The COM

of each injection site was calculated and represented

as single markers to define the locations of mPFC injec-

tion sites along the rostrocaudal axis shown in Figure

1A (see Methods). Figure 1C shows a collapsed repre-

sentation of mPFC labeled layer V tissue for all nine

cases in rat. The mean of the anterior-posterior coordi-

nate of the COM calculated for each injection site

(weighted by each injection’s layer V volume) was

13.12B, with 50% of injected tracer observed between

13.80B and 12.25B (Fig. 1C, left boxplot).

RabbitsThe sites of anterograde tracer injections in the rabbit

(n 5 7) were located 1.0–2.0 mm lateral to the midline,

and were distributed between 15.05B and 10.9B

(Figs. 1B, 3B). Similar to that observed for the rats, the

fluorescent tracer typically spanned all cell layers of

mPFC, diffusing out from the base of the syringe and

was observed along the entire syringe tract, labeling

cells in superficial as well as deep layers of the mPFC.

Using identical procedures as that described for the

rats, the extent and precise location of labeled mPFC

corticopontine neurons in rabbits was restricted to the

volume of layer V tissue in which brightly labeled cells

were observed. Again, the exclusion of gliotic tissue

often resulted in irregularly shaped layer V injection

sites that in the case of the rabbits spanned 300–900

lm across the anterior-posterior extent of the mPFC

(Fig. 3B; median of anterior-posterior extent 5 600 lm).

This increased spread of labeled tissue was likely due

to the increased volume of anterograde tracer injected

for rabbit (1.0 lL versus 0.25 lL). However, even with

the larger injection sites less tissue as a proportion of

total mPFC volume appeared labeled in rabbits relative

to rats. Similar to rats, labeled layer V neurons from a

given injection site in the rabbits often appeared to

span more than one presumed mPFC subregion. The

most common sampled mPFC subregion in rabbit was

the AGm, labeled in 7/7 cases ranging between

15.05B and 10.9B. The AC was labeled in 6/7 cases

across the same anterior-posterior range. The dorsal PL

was labeled in one case in rabbit, between 15.05B and

14.45B. Figure 1C (right) shows a collapsed represen-

tation of mPFC labeled layer V tissue for all seven

cases in rabbit. The mean of the COM calculated for

each injection site (weighted by each injection’s vol-

ume) was 13.48B, with 50% of injected tracer observed

between 14.17B and 12.17B (Fig. 1C, right boxplot).

Comparison between rats and rabbitsThere was substantial overlap of labeled layer V tissue

between rats and rabbits on a relative scale (anchored

to the genu of the corpus callosum) along the rostro-

caudal axis of the mPFC (�75% overlap between the

two species; Fig. 1). Overall, the mPFC injection sites

between rats are rabbits were similar in terms of rela-

tive placement within mPFC (Figs. 1C, 3). The injection

sites between the two species differed in that there

were fewer rabbit cases with more rostral infusion sites,

and in the proportionately larger extent of labeled layer

V tissue in rats given that the rabbit brain is �50%

larger than the rat brain, while the average size of rab-

bit mPFC labeled tissue was only 20% larger. As a

result, there was less complete sampling of the rabbit

mPFC across the rostrocaudal axis compared to that

obtained for the rat.

mPFC terminal labeling in ponsSingle injections of anterograde dextran tracer into

the mPFC of rats and rabbits resulted in multiple dis-

crete clusters of punctate labeling in the pons, repre-

senting putative mPFC labeled axon terminals, that

varied in number and density (e.g., Fig. 4). Relative den-

sity coefficients were determined for each discrete clus-

ter of terminals (e.g., Fig. 4D–G), and were used to

calculate the weighted COM for each system of termi-

nal clusters in the pons observed for a given mPFC

injection site (see Materials and Methods).

RatsThe loci of terminal clusters were primarily observed in

the rostral one-third of the ipsilateral and contralateral

pons in rat, most often in the medial to central region

(Fig. 5A,C, left; four representative examples from indi-

vidual injection sites distributed across the rostral-

caudal axis are given in Fig. 6, 3D representations of all

individual cases are shown in Fig. 8). Six out of nine

cases revealed terminal fields ipsilateral to the mPFC

injection site that were strikingly similar across individu-

als. These projection fields typically extended from the

ventromedial edge of the pyramidal tract to the ventro-

medial surface of the pons, appearing roughly columnar

in shape and ranging between 950 and 1,350 lm in

length, and 620 to 900 lm at maximum width (e.g.,

Fig. 6B–D). These terminal clusters sometimes

extended through the entire rostral half of the pons

(600 lm), and appeared most dense at the rostral

extent. Additional ipsilateral terminal fields were

observed in these and the remaining three cases, and

tended to be smaller and varied in shape, ranging from

nearly spherical clusters with diameters of �250 lm,



to more elongated clusters 580 3 150 3 100 lm in

length, width, and rostral-caudal extent, respectively

(e.g., Fig. 6). Clusters of mPFC axon terminals were typ-

ically most dense in the ipsilateral pons, with contralat-

eral labeling often appearing as a less dense "mirror

image" of a subset of the ispilateral labeled terminals in

7/9 cases (e.g., arrow in Fig. 4C; Fig. 6B,C; Fig. 8

cases 2–7 and 9; contralateral labeling was not

Figure 5. Schematics of the location of the pons and gross anatomical structure in rats and rabbits, and distribution of putative prefrontal

terminals in the pons observed in each species. A: Representation of the rat brain (viewed from the ventral surface) showing the location

of the pons (dark gray) and associated pyramidal tract (py, light gray). Coronal sections and line drawings from the more anterior (top)

and posterior (bottom) pons are also shown (numbers indicate anterior-posterior stereotaxic coordinates relative to Bregma). B: Represen-

tation of the rabbit brain, as described for rats in A. C: Example coronal sections from along the rostrocaudal axis of the pons (numbers

indicate stereotaxic coordinates relative to Bregma). Summation of the extent and density of prefrontal terminal clusters across all cases

for rat and rabbit (shown in red). Boxplots (center) show the COM of labeled terminals (black markers) and IQR (gray box) for rats and rab-

bits. Note the difference in gross anatomical location of the pyramidal tract throughout the pons between rat and rabbit. Prefrontal termi-

nals were preferentially labeled and were densest in the anterior pons of rats, while prefrontal terminals were observed at relatively more

caudal locations surrounding the descending pyramidal tract in the intermediate pons.



observed in two cases, Fig. 8 cases 1 and 8). Contralat-

eral clusters of terminals varied in size and shape, but

tended to be more ellipsoidal than corresponding col-

umns of ipsilateral labeling (they appeared to lack the

dorsal component that was most proximal to the

pyramidal tract). The largest contralateral terminal clus-

ters were �850 lm in length by 300 lm in width by

250 lm in rostral-caudal extent. Smaller clusters of

labeled terminals were �200 3 150 3 100 lm. The

ipsilateral pyramidal tract is assumed to be the source

of contralateral labeling, given that no labeling was

observed in the pyramidal tract contralateral to the

mPFC injection site (e.g., Fig. 4C, double-asterisk).

In 8/9 cases terminal labeling was observed in the

rostralmost 100 lm of the rat pons (Fig. 5C, left; the

exception is shown in Fig. 6E). The COM of terminal

Figure 6. Prefrontal terminal labeling was observed in the rostral pons in all cases for rat, independent of the anterior-posterior coordinate

or subregional location of labeled somata within the medial prefrontal cortex. A: Extent of labeled layer V tissue observed across cases in

rat (bottom line), and the COM (colored markers) and IQRs of prefrontal labeled tissue for four individual cases (top; colors correspond to

individual case assignments shown in 3D renderings in Figs. 8, 10A). B–E: Line drawings and general information (COM, anterior-posterior

spread, Layer 5 volume and prefrontal subregion: AGm, medial agranular; AC, anterior cingulate; dPL, dorsal prelimbic; M1, primary motor

cortex) of labeled layer V tissue (red) in the medial prefrontal cortex (top), and the prefrontal terminal labeling observed for that injection

(below). Anterior-posterior stereotaxic coordinates (relative to Bregma) and COM 6 IQR calculated for each example are given.



clusters in the pons resulting from each mPFC injection

site was located in the rostral 400 lm of the pons in

8/9 cases in the rat (e.g., Fig. 6B–E, black markers).

The IQRs of weighted terminal clusters for these injec-

tions sites were also restricted to the rostral 400 lm

and substantially skewed toward the rostral pole rela-

tive to the COM, indicating that 75% of mPFC terminals

were observed in the rostral pons in rats (e.g., Fig. 6B–

E, boxplots; 3D data shown in Fig. 8 for all individual

cases and cumulatively in Fig. 10A). An exception to

this pattern was observed in three cases, which in addi-

tion to dense rostral labeling also displayed a relatively

small but dense region of terminal labeling that was

typically restricted to the medial wall and ventral aspect

of the pons along nearly the entire rostrocaudal extent

of the structure (Fig. 8, cases 2–4). The only difference

noted between the injection sites in these versus other

cases was labeling at or near the dorsal PL – ventral

AC border. Interestingly, there were no discernable dif-

ferences in the patterns of putative terminals in the

pons resulting from rostral versus caudal mPFC injec-

tion sites in the rat—the pons labeling from more rostral

mPFC infusions were remarkably similar to that

observed for more caudal infusion sites (e.g., Figs. 6,

8). Additionally, no systematic differences in mPFC cor-

ticopontine projection patterns were observed between

anterograde tracer injections in the different subregions

of mPFC sampled in this study, with the possible excep-

tion described above.

RabbitsIn contrast to rats, the loci of clusters of mPFC termi-

nals was observed in the intermediate region of the

ipsilateral pons of the rabbit, most often in close asso-

ciation with the pyramidal tract as it descends ventrally

and medially through the pons (Fig. 5C, right, and Figs.

9, 10B). Terminal clusters in the rabbit were often more

elliptical, in further contrast to the columnar shape of

the most dense mPFC terminal clusters in the rat pons.

In 5/7 cases the largest and densest clusters of termi-

nals were observed in the areas immediately medial or

lateral to the ventral pyramidal tract (Figs. 5C, left,

Fig. 7). The dimensions of these mPFC terminal clusters

ranged from 500–1,100 lm in length and 450–700 lm

in width, and typically extended 400–750 lm in the

anterior-posterior axis. Smaller clusters of varying den-

sity were also observed distributed throughout the

mediodorsal and lateral pons. These clusters ranged in

size from 150–180 lm and 300–250 lm in length and

width, respectively, and extended 250–500 lm in the

rostral-caudal axis. Although single axons were occa-

sionally observed exiting the pyramidal tract and cross-

ing the midline to the contralateral hemisphere,

clusters of labeled terminals were not observed in the

contralateral rabbit pons for any of the mPFC injections

(Fig. 7B–E; Figs. 9, 10B).

Clusters of mPFC terminals were not observed in the

most rostral 800 lm of the rabbit pons (Fig. 5C, left).

Instead, mPFC terminals were restricted to the interme-

diate pons in 7/7 cases, and typically showed clusters

of terminals that were distributed symmetrically around

the COM along the rostral-caudal axis (Fig. 7B–E, black

markers and boxplots). Patterns of pontine terminal

labeling were not different between caudal and rostral

mPFC injection sites in rabbit, similar to the finding in

rat (compare Fig. 7B,C to 7E; Fig. 9). Likewise, no sys-

tematic differences in projection patterns were

observed between anterograde tracer injections in the

different subregions of mPFC sampled in this study.

Comparison of mPFC projection patternsbetween rats and rabbitsA collapsed summary of all labeled terminals observed

in the pons for rat and rabbit is given in Figure 5C and

a 3D summary in Figure 10. The loci of the clusters of

mPFC terminals from all rats was biased toward the

rostral pole of the rat pons, while the rostral pons of

rabbits was devoid of mPFC terminal clusters, even

though there was substantial overlap in the relative

mPFC regions of anterograde tracer uptake by layer V

cells between rats and rabbits (Figs. 1, 3, 10). Some

mPFC terminal labeling was observed in the intermedi-

ate pons of rats that overlapped with the locus of ter-

minal labeling in the rabbit pons, but comprised less

than 25% of all mPFC terminal clusters in the rat pons

(Fig. 5C). In Figure 10, species-specific prefrontal termi-

nal labeling in the medial pons can be compared when

each species is viewed from an anterior perspective

(Pons, top image), while differences in terminal labeling

in the rostral pons is best noted from a ventral perspec-

tive (Pons, bottom image). A striking difference in

mPFC corticopontine projections between the two spe-

cies is the substantial contralateral labeling observed in

most cases in rat, which was completely absent in rab-

bit (Figs. 5–10).

DISCUSSION

The patterns of mPFC projections to the pons were

characterized for rat and rabbit using anterograde trac-

ing techniques. There were striking differences in the

distribution of labeled prefrontal axon terminals

between the two species. Both the overall topography

of projections across the rostral-caudal axis and the

degree of contralateral labeling observed differed

between rats and rabbits.



Species-specific differences in therostral-caudal distributions of mPFCcorticopontine terminals

More than 75% of mPFC labeled terminals in rat

were observed in the rostral half of the medial pons,

often extending from the ventromedial pyramidal tract

toward the ventral surface of the pons (Figs. 5C, left,

and 10A). In contrast, the rostral pons of rabbit was

entirely devoid of mPFC labeled terminals (Figs. 5C,

right, and 10B). Instead, mPFC corticopontine terminals

in rabbit tended to surround the pyramidal tract as it

descended ventromedially through the pons over the

intermediate rostral-caudal extent (Fig. 10).

It is unlikely that the species-specific divergence in

mPFC corticopontine projections demonstrated here

could be explained by potential anterior-posterior differ-

ences in the coordinates of the mPFC injection sites

between the rats and rabbits used for this study. First,

Figure 7. Prefrontal terminal labeling was observed in the intermediate to more caudal pons in all cases for rabbit, independent of the

anterior-posterior coordinate or subregional location of labeled somata within the medial prefrontal cortex. A: Extent of labeled layer V tis-

sue observed across cases (bottom line), and the COM (colored markers) and IQRs of prefrontal labeled tissue for four individual cases

(top; colors correspond to individual case assignments shown in 3D renderings in Figs. 9, 10B). B–E: Line drawings and general informa-

tion (as described in Fig. 6) of labeled layer V tissue (red) in the medial prefrontal cortex (top), and the prefrontal terminal labeling

observed for that injection (below). Anterior-posterior stereotaxic coordinates (relative to Bregma) and COM 6 IQR calculated for each

example are given.



the locations of mPFC injection sites in the rats and

rabbits overlapped considerably in a relative anterior-

posterior axis (by �75%; Figs. 1, 3), while the overlap

of mPFC terminals in the pons showed very little over-

lap (<25%; Figs. 5C, 10). Second, the species-specific

differences are still observed when only mPFC injec-

tions within the same relative rostral-caudal extent are

considered (injections between 11.0–3.5B in rats and

Figure 8. Three-dimensional reconstructions of labeled layer V tissue resulting from tracer injection in mPFC and associated terminal label-

ing in the pons for each rat. Cases are ordered from most rostral to the most caudal prefrontal injection site (as columns running from

top to bottom). Two perspectives are shown for each injection site (PFC, left) and for each reconstructed pons (right). Directional com-

passes as described in Figure 2. The patterns of terminal labeling in the rostromedial pons were not different across cases depending on

anterior-posterior location of prefrontal injection sites. Terminal labeling in the medial pons across cases can be compared when viewed

from an anterior perspective (Pons, top image), while terminal labeling in the rostral pons across cases is best noted from a ventral per-

spective (Pons, bottom image).



12.0–5.0B in rabbits, Fig. 3; compare individual rats

from Fig. 6C–E to individual rabbits given in Fig. 7C,D).

Finally, injections made at various locations along the

anterior-posterior extent of the mPFC did not reveal dis-

tinct patterns of pontine terminal labeling within spe-

cies (Fig. 6B–E for rat, Fig. 7B–E for rabbit), and so any

rostral-caudal differences in the injection sites between

the rats and rabbits is unlikely to explain the species-

specific results. Furthermore, data from previous stud-

ies in which more caudal rat mPFC injections or more

Figure 9. Three-dimensional reconstructions of labeled layer V tissue from prefrontal (PFC) tracer injection and associated terminal label-

ing in the pons for each rabbit. Cases are ordered from most rostral to the most caudal prefrontal injection site (as columns running from

top to bottom). Two perspectives are shown for each injection site (PFC, left) and for each reconstructed pons (right). Directional com-

passes as described in Figure 2. The patterns of terminal labeling in the intermediate to caudal pons were not different across cases

depending on anterior-posterior location of prefrontal injection sites. Terminal labeling in association with the descending pyramidal tract

is clearly observed from both anterior (Pons, top image) and ventral perspectives (Pons, bottom image).



rostral rabbit mPFC injections resulted in similar

patterns of pons terminal labeling as reported here

(Wiesendanger and Wiesendanger, 1982b; Buchanan

et al., 1994). Together, the evidence strongly suggests

that differences in the pattern of mPFC projections

reported here between rat and rabbit represent a

species-specific divergence in the topography of corti-

copontine projection patterns.

Species-specific differences in contralateralmPFC corticopontine projections

This comparative study revealed contralateral projec-

tions from mPFC to the pons in rat that were not

observed in rabbit. Discrete clusters of labeled mPFC

terminals were observed in the rostromedial region of

the contralateral pons in rat, often mirroring the pattern

of terminal clusters observed on the ipsilateral side. In

contrast, clusters of labeled terminals were never

observed in the contralateral pons of rabbit (Figs. 5, 7,

9, 10). It should also be noted that labeled axons were

never observed in the contralateral pyramidal tract, so

it is presumed that labeled axons from the ipsilateral

pyramidal tract crossed the midline and formed termi-

nal clusters in the contralateral pons in the rat. Given

the absence of labeled axons in the contralateral

pyramidal tract, it is unlikely that uptake of the antero-

grade tracer by callosally projecting fibers could have

resulted in apparent contralateral labeling. In the rabbit,

single axons were occasionally observed crossing the

Figure 10. Summary of medial prefrontal labeled layer V tissue resulting from injection of anterograde label, and the pattern of putative

terminal labeling in the pons in rat (A) and rabbit (B). Three-dimensional perspectives and compass directions are as described for Figures

8 and 9. Note the species-specific patterns of prefrontal terminal labeling observed between rat and rabbit even though overlapping

regions of medial prefrontal cortex were labeled for the two species. The medial prefrontal cortex of rats preferentially projects to the ros-

tromedial pons, while the mPFC of rabbits preferentially projects to pontine regions that surround the pyramidal tract as it descends ven-

tromedially through the intermediate to more caudal pons.



midline but did not give rise to terminal clusters in the

contralateral pons. It is possible that these axons con-

tributed to very sparse or dispersed contralateral termi-

nal labeling in rabbit that was not detected by our

analysis. However, the presence of diffusely labeled ter-

minals in the contralateral pons of rabbit would still be

in sharp contrast to the readily apparent terminal clus-

ters observed in the contralateral pons of rat.

Qualifications for the current studyThe proportion of labeled tissue relative to mPFC vol-

ume appeared smaller in the rabbit, despite the fact

that considerably more tracer was injected. This would

likely still be true even if our analysis underestimated

the volume of labeled tissue at the injection sites.

Therefore, proportionally less of the mPFC was sampled

in rabbit than in rat, which included unsampled regions

within the rostrocaudal extent (Fig. 3). Therefore, it is

possible that unsampled mPFC regions in rabbit may

have shown rostral or contralateral labeling in the pons.

This seems unlikely, given that rostral and caudal

extents of the same mPFC subregions showed largely

overlapping and entirely ipsilateral terminal labeling, but

cannot be ruled out in this study.

Comparison of findings to previous workThe current study both supported and further refined

previous work examining the specific pattern of pontine

projections from the mPFC in rats and rabbits by using

more spatially restricted anterograde tracer injections,

and by further defining injection sites relative to labeled

layer V tissue. Furthermore, we allowed for direct com-

parisons between rat and rabbit, and revealed substan-

tial species-specific differences in the topography of

mPFC corticopontine projection patterns. The pattern of

mPFC to pons projections demonstrated here is in gen-

eral agreement with the overall topography of inputs

from frontal cortical regions to the rostromedial pons

previously reported for rat (Wiesendanger and Wiesen-

danger, 1982a,b; Leergaard and Bjaalie, 2007). More

specifically, the relative restriction of mPFC terminals to

the rostral half to one-third of the medial pons, in addi-

tion to distinct contralateral labeling, was also reported

in prior studies (Domesick, 1969; Wiesendanger and

Wiesendanger, 1982b; Wyss and Sripanidkulchai, 1984;

Leergaard and Bjaalie, 2007). The overall pattern of

mPFC to pons projections in rat is similar to that dem-

onstrated for monkey (Vilensky and Van Hoesen, 1981;

Schmahmann and Pandya, 1997b) and cat (Brodal and

Bjaalie, 1992), and is in sharp contrast to that observed

for rabbit. We noted mPFC terminal fields immediately

surrounding the pyramidal tract as it descended ventro-

medially through the intermediate anterior-posterior

extent of the pons in rabbit, which were not observed

in the rostral or contralateral pons. Although early

reports showed frontal cortical terminal fields restricted

to the dorsal and medial peduncular pons in rabbit

(Abdel-Kader, 1968), the pattern of mPFC inputs

reported here is consistent with more recent investiga-

tions that focused on corticopontine inputs specifically

from the mPFC (Buchanan et al., 1994; Weible et al.,

2007; Siegel et al., 2012).

Similarities in corticopontine projectionsbetween rat and rabbit

The ability to localize injection sites to layer V cells

(the well-established source of corticopontine inputs;

e.g., Legg et al., 1989) allowed for more precise local-

ization of mPFC cells that were the source of terminal

labeling in the pons. Interestingly, mPFC injection sites

at different anterior-posterior coordinates did not reveal

distinct mPFC corticopontine projection patterns within

species (i.e., compare Fig. 6B–E across rats, compare

Fig. 7B–E across rabbits). Similarly, there were no con-

sistent differences in the location of labeled terminals

between the mPFC subregions sampled here within

either species. This was also true for the anterior and

posterior extents of the same mPFC subregion, particu-

larly rostral and caudal AC (rat: compare injection sites

from rat 1, Fig. 8; rabbit: compare injection sites from

rabbit 3, Fig. 9), which the data suggest can play differ-

ent functional roles (Kronforst-Collins and Disterhoft,

1998; Hattori et al., 2010). An exception may exist in

rat for the two most caudal injections (between 12.0

and 11.35B, one of which is shown in Fig. 6E). Medial

PFC projections were not observed at the rostralmost

pole in these two cases, and showed little if any contra-

lateral labeling. Although previously reported for rat

(Domesick, 1969; Wyss and Sripanidkulchai, 1984), the

generally indistinguishable patterns of mPFC corticopon-

tine terminal labeling across these dimensions within

species is particularly compelling given the highly

restricted and spatially segregated injection sites in the

current study, even if separated by 2 mm or more in

the anterior-posterior axis.

An additional similarity between rat and rabbit was

that for individual injection sites, we observed multiple

discrete patches of mPFC labeled terminals distributed

over a restricted pontine region. This topographically

constrained yet highly divergent pattern is similar to

previous reports of corticopontine projections in all spe-

cies studied (Abdel-Kader, 1968; Wiesendanger and

Wiesendanger, 1982b; Brodal and Bjaalie, 1992; Leer-

gaard and Bjaalie, 2007). It has been speculated that

each patch represents a "private" or isolated line of



input from a fairly restricted area of cortex, and that

the distribution of patches from a given input source

serves to ensure that the information from the cortical

input source reaches expansive regions of cerebellar

cortex (reviewed in Wiesendanger and Wiesendanger,

1982b; Brodal and Bjaalie, 1992). More controversial is

the degree to which inputs near the borders of adjacent

terminal patches may converge onto the same pontine

neurons, the output of which would reflect some inte-

gration of those inputs (Wiesendanger and Wiesen-

danger, 1982b; Kosinski et al., 1988; Brodal and

Bjaalie, 1992; but see Schwarz and M€ock, 2001;

Schwarz et al., 2005). Putative convergence of cortico-

pontine inputs has been substantiated with double-

anterograde tracer studies in cat demonstrating some

partial overlap of adjacent terminal fields from distinct

cortical regions (Bjaalie and Brodal, 1989; Brodal et al.,

1991). The functional integration of inputs from differ-

ent cortical regions has been suggested by single-unit

electrophysiological studies in cat, monkey, and rat

demonstrating the ability to evoke spike activity when

stimulating distinct but nearby cortical regions in nearly

half of sampled pontine projection cells (R€uegg and

Wiesendanger, 1975; R€uegg et al., 1977; Potter et al.,

1978). Although the data presented here do not

address this issue, its consideration is essential in order

to determine the possible impact of the different topog-

raphies of mPFC to pons projection patterns observed

between rat and rabbit. However, it should be noted

that if a complete lack of convergence between adja-

cent terminal fields in the rat and rabbit pons were to

be demonstrated it would suggest that the general role

of the pons would be more similar to that of a relay

station. Such a finding presumes that pontine neurons

would simply pass on information regarding upstream

spike activity without any integration of those inputs,

making the exact topography of corticopontine projec-

tions of little functional relevance. Even so, the species-

specific differences reported here would continue to be

of considerable interest from a developmental perspec-

tive (O’Leary et al., 1991; Leergaard et al., 1995).

Functional impact of species-specificdifferences in corticopontine projections

It is hypothesized that any integration of inputs in

the pons would only occur at the putative overlapping

borders of adjacent terminal fields. Thus, species-

specific differences in corticopontine projection pat-

terns could result in the integration of different cortical

inputs depending on what other brain areas project to

the same region of the pons in that species. It should

be noted that the general corticopontine mapping

described for various species is an acknowledged over-

simplification, with terminal patches also commonly

observed outside of the general projection region (Wie-

sendanger and Wiesendanger, 1982b; Leergaard and

Bjaalie, 2007). Nevertheless, the broad topographical

organization of corticopontine inputs established for rat

predicts that the greatest opportunity for integration

with mPFC inputs would occur with input from rostro-

medial primary motor areas (such as frontal eye field,

whisker, and potentially forelimb primary and supple-

mentary motor cortex regions; Wiesendanger and Wie-

sendanger, 1982a; Kosinski et al., 1986; Guandalini,

2001; Alloway et al., 2010) and the rostralmost subre-

gions of primary somatosensory cortex (such as barrel

cortex and possibly forelimb; Wiesendanger and Wie-

sendanger, 1982a; Kosinski et al., 1986; Alloway et al.,

2010). Corticopontine input from auditory, visual, retro-

splenial, and caudal or lateral somatosensory regions

would be least likely to be directly integrated with

mPFC input given that the terminal fields from those

structures tend to lie far lateral or in the caudal half of

the pons in rat (Wiesendanger and Wiesendanger,

1982a,b; Azizi et al., 1985; Schwarz et al., 2005; Leer-

gaard and Bjaalie, 2007).

Although a broad topographical organization for rab-

bit corticopontine projections is far less established,

the available data suggest similarities with rat in that

there would be the highest opportunity for integration

with primary motor regions, and less so for auditory

and visual cortical input (Abdel-Kader, 1968; Wells

et al., 1989; Knowlton et al., 1993). A notable distinc-

tion between the potential for the integration of inputs

in the pons between rat and rabbit is the apparent

topographical overlap of mPFC terminal fields in rabbit

with input from somatosensory and retrosplenial corti-

ces (according to the map of Abdel-Kader, 1968). A

second distinction is the putative overlap of mPFC corti-

copontine inputs with that of the deep layer, multi-

modal region of the superior colliculus in rabbit (Wells

et al., 1989), while in rat inputs from the deep layers of

the super colliculus are restricted to the caudolateral

pons (Burne et al., 1981) and do not appear to overlap

topographically with mPFC inputs in the rat pons. With

the exception of auditory and visual corticopontine

inputs, it appears that mPFC inputs in rabbit tends to

violate a confined topographical projection pattern in

that it surrounds and extends from the descending

pyramidal tract, while other cortical inputs are reported

to remain more restricted within their respective dorsal,

lateral, or ventromedial borders relative to the

peduncle. Overall, the available data suggest that mPFC

corticopontine input in rabbit is generally more likely to

be integrated with input from somatosensory and asso-

ciational brain regions than would be predicted in rat.



Implications of species-specific differencesin prefrontopontine projections for traceeyeblink conditioning

The primary motivation for the current study was to

investigate the possibility of species-specific differences

in prefrontopontine input patterns between rabbit and

rat as it relates to trace eyeblink conditioning. The

mPFC and cerebellum have been shown to be neces-

sary for the expression of trace conditioned responses

in both species (e.g., Kronforst-Collins and Disterhoft,

1998; Takehara et al., 2003; Kalmbach, 2008), with

prefrontal inputs reaching the cerebellum via the pons.

In rats, experiments focusing on the mPFC would likely

require bilateral manipulation, assuming that both

Figure 11. Schematic of prefrontocerebellar projection pathway in rats (left) and rabbits (right), with colors indicating projection sites for

the subregions of mPFC throughout the pathway (green: medial agranular, blue: anterior cingulate, red: prelimbic). Note that the rostral

and caudal extents of a given subregion projected to largely overlapping pontine regions (see text). In rat, the current study demonstrates

that mPFC subregions project to the ipsilateral and less densely to the contralateral rostromedial pons via the ipsilateral pyramidal tract.

Previous work has shown that the ipsilateral and contralateral pons each project predominantly to the opposite cerebellar hemisphere and

modestly to the same hemisphere, including the anterior portion of Lobules V/VI that support eyeblink conditioning (see text). In rabbit,

the mPFC projects ipsilaterally to the intermediate/caudal peduncular pons, which then projects predominantly to the contralateral cere-

bellar hemisphere and modestly to the same hemisphere, including cerebellar cortex regions that support eyeblink conditioning. Note that

pontocerebellar projections in both species actually project expansively across cerebellar lobules, and are not indicated here (Brodal and

Bjaalie, 1992).



hemispheres would receive stimulus-associated inputs

from presentation of a tone or light CS. Because mPFC

inputs to the pons are unilateral in rabbits, bilateral

manipulation should be unnecessary. Clearly, different

regions of the pons would be predicted to support trace

conditioning in rats and rabbits based on the current

study. As such, manipulations of mPFC inputs to the

cerebellum should be bilateral and focused on the ros-

tromedial pons in rats, and in the intermediate to cau-

dal peduncular region unilaterally in rabbits (Fig. 11). A

putative role for the rostromedial pons in trace eyeblink

conditioning has not been tested in rat, but unilateral

infusions of muscimol in the intermediate lateral pons

in rabbit, located just adjacent to the peduncle, can

abolish trace conditioned responses (Kalmbach et al.,

2009).

Projections from the pons to the cerebellum are pre-

dominantly, but not exclusively, to the contralateral cer-

ebellar hemisphere in both species (Mihailoff, 1983;

Yeo et al., 1985; Serapide et al., 2001; Tracy et al.,

2013). Importantly, the rostromedial pons in rat and

the intermediate peduncular pons in rabbit both include

projections to cerebellar lobules V/VI (Fig. 11; Mihail-

off, 1983; Yeo et al., 1985b; Serapide et al., 2001;

Tracy et al., 2013), which are the established regions of

cerebellar cortex shown to support eyeblink condition-

ing in rabbits and rats (Yeo et al., 1985a; Garcia et al.,

1999; Plakke et al., 2007; Kalmbach et al., 2010).

Because eyeblink conditioning does not require bilateral

coordination (only one eye is typically trained) there is

likely little implication of unilateral versus bilateral

inputs at the level of the cerebellum for this learning

paradigm, except perhaps under specific circumstan-

ces. For example, if after acquisition training is

switched to the opposite eye, one might expect accel-

erated learning because the opposite cerebellar hemi-

sphere would not be na€ıve to the CS. In support, Yeo

et al. (1985a) found that post-learning lesions of cere-

bellar lobule HVI abolished CRs, but that rabbits

relearned quickly when the opposite eye was trained

with the same CS. However, eyeblink conditioning could

be used to test the functional nature of the prefronto-

pontine contralateral projection in rat using microstimu-

lation of the mPFC ipsilateral to the trained eye as the

CS (learned behavior would be mediated by the ipsilat-

eral cerebellum, which under these conditions would

only receive the more modest prefrontal inputs to the

contralateral pons). Because the prefrontocerebellar

input is bilateral, rats should be able to learn independ-

ently of which hemisphere is stimulated, while the uni-

lateral nature of the prefrontocerebellar pathway in

rabbits should preclude learning if the ipsilateral mPFC

is stimulated.

CONFLICT OF INTEREST

The authors state no identified conflict of interest

regarding the current work.

AUTHOR CONTRIBUTIONS

All authors had full access to all the data in the

study and take responsibility for the integrity of the

data and the accuracy of the data analysis, and have

read and revised the submitted article. Study concept

and design: JJS, RAC, ND, BEK, MVM. Acquisition of

data: JJS, RAC, MVM, EDM. Analysis and interpretation

of data: RAC, JJS, MVM, EDM. Drafting of the article:

JJS, MVM, RAC. Statistical analysis: JJS, MVM. Adminis-

trative, technical, and material support: EDM. Project

supervision: RAC, JJS, and DJ.

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